Invitrogen™

DNA Polymerase I

Name: DNA Polymerase I
SKU: 18010025
Price: 872.00 EUR

L’ADN polymérase I est un ADN polymérase présentant 5´→3´ et 3´→5´ activités d’exodésoxyribonucléase. L’ADN polymérase I intègre également des nucléotidesAfficher plus

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Référence	Quantité
18010025	1000 U
18010017	250 U

2 options

Référence 18010025

Prix (EUR)

872,00

Each

Quantité:

1000 U

Prix (EUR)

872,00

Each

L’ADN polymérase I est un ADN polymérase présentant 5´→3´ et 3´→5´ activités d’exodésoxyribonucléase. L’ADN polymérase I intègre également des nucléotides biotinylés.

• Applications—Traduction de nick dépendante de la DNase I, synthèse de second brin dans le clonage de l’ADNc, remplissage de 5´ en suspension
• Source—purifiée à partir d’E. coli exprimant le gène de l’ADN polymérase I sur un plasmide

Test de performance et de qualité
Matrice d’endodésoxyribonucléase ; efficacité de la traduction de nick dépendante de la DNase I déterminée.

Définition d’unité
Une unité intègre 10 nmol de désoxyribonucléotide total dans du matériau précipitable par acide en 30 min à 37°C à l’aide d’un modèle/d’une amorce.

Conditions de réaction de l’unité :
50 mM de phosphate de potassium (pH 7,0), 6,7 mM de MgCl₂, 1 mM de 2-mercaptoéthanol, 80 µg/ml de modèle/amorce, 32 µM de dTTP, 69 nM [³H] de dTTP et enzyme dans 100 µL pendant 30 min à 37°C.

Usage exclusivement réservé à la recherche. Ne pas utiliser pour des procédures de diagnostic.

Spécifications

DescriptionADN polymérase

Activité exonucléasique3' - 5', 5' - 3'

Hot StartNon

PolyméraseDNA Polymerase I

Quantité1000 U

Conditions d’expéditionGlace humide

Unit SizeEach

Contenu et stockage

1 000 unités d'ADN polymérase dans un tampon de stockage de phosphate de potassium 50 mM (pH 7,0), de KCl 100 mM, de DTT 1 mM et de glycérol 50 % (v/v). Il est fourni à 5–10 unités/µl.

Stocker à -5°C à -30°C.

Foire aux questions (FAQ)

Is there anything to prevent AmpliTaq Gold DNA polymerase from extending from the 3’ end of a TaqMan probe in a 5’ nuclease assay?

Yes. There is a phosphate group on the 3' end of all TaqMan probes that prevents such extension.

How does AmpliTaq Gold DNA Polymerase differ from AmpliTaq DNA Polymerase?

AmpliTaq Gold DNA Polymerase is a modified form of AmpliTaq DNA Polymerase that contains a proprietary chemical (or so-called hot start molecule) bound to the enzyme's active site. In order to activate the AmpliTaq Gold DNA Polymerase fully, we recommend an initial activation step of 95 degrees C for 10 min when using GeneAmp 10X PCR Buffer I and/or GeneAmp 10X PCR Buffer II and Mg in one of our thermal cyclers. When using GeneAmp 10X PCR Gold Buffer, activation time can be reduced to 5 minutes. Once activation is complete, you can proceed with your standard PCR cycling program (denaturing, annealing, extension, etc).

Does AmpliTaq Gold DNA Polymerase contain exonuclease (proofreading) activity?

No, AmpliTaq Gold DNA polymerase does not contain proofreading activity, however fidelity in PCR amplifications utilizing this enzyme may be improved. High fidelity can be achieved by: 1. Decreasing the final concentration of each nucleotide to 40-50 uM. 2. Using the lowest MgCl2 concentration possible. 3. Using less enzyme. 4. Decreasing extension times. 5. Using the highest annealing temperature possible. 6. Using as few cycles as possible.

Does the fidelity of AmpliTaq DNA Polymerase change in the presence of base analogs?

The fidelity of this PCR enzyme is affected in two ways. First, AmpliTaq DNA Polymerase typically binds to and incorporates base analogs less efficiently than conventional dNTPs, which means that polymerase activity is lower in reactions that contain base analogs. Second, the analog may pair with more than one conventional complementary template base, so the analog may be incorporated at an increased level compared to conventional dNTPs. For the best fidelity, we recommend that base analogs are included at low concentrations in the reaction.

What is the expected half life of AmpliTaq DNA Polymerase at 95 degrees C?

The half-life of AmpliTaq DNA Polymerase at 95 degrees C is 40 min. During PCR, the sample is only incubated at the programmed temperature for approximately 20 seconds. Therefore, the cycling half-life of AmpliTaq Gold at 95 degrees C is approximately 100 cycles.

Example: AmpliTaq DNA Polymerase experiences about 20 seconds at 95 degrees C per PCR cycle. The t_1/2 is at least 33 minutes; (35-40 min). Therefore, 33 min/20 sec/cycle = 100 cycles. 100 PCR cycles reduces enzyme activity by 50%.

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